7-chloro-6-demethyl-tetracycline fermentation

ABSTRACT

This disclosure describes a process for the production of 7chloro-6-demethyltetracycline to the exclusion of 6demethyltetracycline by fermentative biosynthesis using strains of Streptomyces aureofaciens which are characterized by their ability to impart to a 1:200 aqueous dilution of the whole harvest mash a color characterized by a spectrophotometric reflectance curve such that when the percent reflectance is plotted linearly against the wavelength the resulting curve, between 460 m Mu and 520 m Mu , will either exhibit a maximum or have a point of inflection of zero slope.

United States Patent [72] Inventor John Andrew Growich, Jr.

New City, N.Y.

[21] Appl. No. 601,250

[22] Filed Oct. 12, 1966 [45] Patented Oct. 26, 1971 [73] Assignee American Cyanamid Company Stamford, Conn.

Continuation-impart of application Ser. No. 508,356, Nov. 17, 1965, now abandoned.

[54] 7-CHLORO-6-DEMETHYL-TETRACYCUNE FERMENTATION 2 Claims, 12 Drawing Figs.

[52] U.S. Cl 195/80 [51] Int.Cl C12d 9/00 [50] Field of Search 195/80 [56] References Cited UNITED STATES PATENTS 2,878,289 3/1959 McCormick et al. 195/80 X Primary Examiner-Joseph M. Golian Attorney-Edward A. Conroy, Jr.

ABSTRACT: This disclosure describes a process for the production of 7-chloro-6-demethyltetracycline to the exclusion of 6-demethyltetracycline by fermentative biosynthesis using strains of Streptomyces aureofaciens which are characterized by their ability to impart to a 1:200 aqueous dilution of the whole harvest mash a color characterized by a spectrophotometric reflectance curve such that when the percent reflectance is plotted linearly against the wavelength the resulting curve, between 460 my and 520 mu, will either exhibit a maximum or have a point of inflection of zero slope.

PATENTEDUBT 26 |97| SHEET UlOF 12 FIG?! REFLECTANCE CURVE OF WHOLE HARVEST M OBTAINED WITH 8. AUREOFACIENS STR ASH AIN S604 WAVELENGTH (MILLIMICRONS) INVENTOR. JOHN ANDREW GRO ;CH,JR.

ATTORNEY PATENTEUUCT 2s l97l PERCENT REFLECTANCE SHEET OEUF 12 REFLEGTANCE CURVE 0F WHOL VEST MASH OBTAINE lTH STRAIN SIO7| WAVELENGTH (MILLIMICRONS) INVENTOR.

ATTORNEY JOHNDREW |CH,JR.

PATENTEBUBT 25 I97! SHEET 03 HF 12 O O m 5 m 5 mozqkom lwm Fw -momma WAVELENGTH (MELLBMUCRONS) INVENTOR.

ATTORNEY PATENTEDum 26 I97! PERCENT REFLECTANCE SHEET CQUF 12 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH AUREOFACIENS STRAIN B740 WAVELENGTH (MILLIMICRONS) INVENTUR.

JOHN ANDREW GROWICH,JR.

ATTORNEY PAIENTEUum 2s l97| PERCENT REFLECTANCE SHEET USUF 12 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH AUREOFACIENS STRAIN Vl38 WAVELENGTH (MILLIMICRONS) INVEN'IUR.

JOHN ANDREW GROWICH,JR.

ATTORNEY PATENTEIJBEI 26 I97! 3,616,240

SHEET UBUF 12 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH s UREI STRAIN E475 PERCENT REFLECTANCE WAVELENGTH (MILLIMICRONS) INVENTOR.

JOHNNDREW GRO CHJR.

BY A

ATTORNEY PATENTEDUBT 26 I9?! 3, E5 1 6,240

SHEET 07UF 12 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH i UEO STRAIN El3ll PERCENT REFLECTANCE WAVELENGTH (MILLIMICRONS) INVENTOR. JOHN ANDREW GROWICH,JR.

ATTORNEY PATENTEBum 26 um PERCENT REFLECTANCE SHEET OBOF 12 REFLECTANCE CURVE OF WHOLE HARVEST MASH OTAINED WITH i STRAIN EDI723 WAVELENGTH (MILLEMICRONS) INVENTOR.

ATTORNEY FATENTEDUBT 26 197i SHEET OSUF 12 FIGrQ REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH 5. AUREQFACIENS STRAIN E0204? uoz kom4mmm hzwomum WAVELENGTH (MILLIMICRONS) INVENTUR.

JOHN ANDREW GROW CH,JR.

A TTORNEY PATENTEDum 26 I9?! 3, 6 1 6 240 SHEET lUUF 12 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED ITH s STRAIN ED23|4 PERCENT REFLECTANCE WAVELENGTH (MILLIMIGRONS) INVENTOR. JOHN ANDREW GROWICH,JR.

ATTORNEY PATENTEDUCT 2 6 I9?! PERCENT REFLECTANCE SHEET 11 0F 12 EFEE REFLECTANG GRVE 0F WHQLE ARVEST MASH oamamm WHTH g STAIN IE2322 WAVELENGTH-9 (MELLEWGRONS) INVENTOR.

JOHN ANDREW GROWICH,JR.

ATTORNEY PATENTEDDN 26 I97\ PERCENT REFLECTANCE SHEET 12 0F 12 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH STRAIN |E275O WAVELENGTH (MflLLIMICRONS) INVENTOR.

JOHN ANDREW GROWICH,JR.

ATTORNEY 7-CHLOR0-6-DEMETHYL-TETRACYCLINE FERMENTATION This application is a continuation-in-part of my copending application Ser. No. 508,356, filed Nov. 17, 1965, now abandoned.

This invention relates to an improved process for the production of 7-chloro-6-demethyltetracycline and, more particularly, is concerned with the production of 7-chloro-6- demethyltetracycline to the exclusion of o-demethyltetracycline by fermentative biosynthesis using strains of Streptomyces aureofaciens which are characterized by their ability to impart to a 1:200 aqueous dilution of the whole harvest mash a color characterized by a spectrophotometric reflectance curve such that when the percent reflectance is plotted linearly against the wavelength, the resulting curve, between 460 my. and 520 my, will either exhibit a maximum or have a point of inflection of zero slope.

The novel mutant strains of the present invention are strains of the species Streptomyces aureofaciens. The representative S. aureofaciens strains described below are direct descendants of the 7-chlorotetracycline-producing S. aureofaciens A-377 soil isolate described in U.S. Pat. No. 2,482,055 to Duggar and deposited at the Northern Regional Research Laboratories, Peoria, ill. and indexed as NRRL 2209, derived by mutagenic processes known to the art, e.g., ultraviolet irradiation, nicotine, and nitrogen mustard. Typical mutant strains of S. aureofacien: which possess the unique properties described herein have been designated by me as -2322 and lE-2750. Viable representative cultures of the novel mutant strains of the present invention have been deposited at the Northern Regional Research Laboratories, Peoria, ill. and have been assigned the following accession numbers:

Strain NRRL No.

lE-2322 3204 IVE-2750 3234 It will be understood, of course, that mutants which possess this unique property of imparting a specific color to their whole harvest mashes may be derived from these strains by conventional procedures. These mutants may vary somewhat in general morphological and physiological characteristics as do the various strains of the S. aureofaciens species. it is also to be expected that 7-chloro-6-demethyltetracycline-producing strains of S. aureofaciens of the invention, i.e., with a reflectance curve having either a maximum or a point of inflection of zero slope between 460 my. and 520 mu, may be found in nature or derived from presently isolated strains S. uUI'EOfGCiEIlS by mutagenic methods well known to the art. The strains of the present invention display the fundamental characteristics of the species S. aureofaciens and may be readi ly identified as members of this species although differing from previously described strains of S. aureofaciens, not only in pigmentation on solid media, but also in the colors of the whole harvest mashes obtained therewith.

The antibiotic 7-chloro-6-demethyltetracycline was first described and claimed, together with methods of its production, in the McCormick et al. U.S. Pat. No. 2,878,289 wherein a 7-chloro-b-demethyltetracycline-producing strain of S. aureofaciens was cultivated in a fermentation medium. That patent also discloses that other tetracyclines, such as 6- demethyltetracycline, 7-chlorotetracycline and tetracycline are produced at the same time in a fermentation medium when the fermentation conditions, including the micro-organism therein disclosed, are used. Additionally, that patent discloses that 7-bromo-6-demethyltetracycline is a concomitant product when bromine is a constituent of the fermentation medium. Further evidence of the production of more than one tetracycline in a fermentation medium in which demethylchlortetracycline is produced may be found in the Perlman et al. U.S. Pat. No. 3,028,3ll and the Neidleman U.S. Pat. No. 3,061,552 wherein so-called 6-methylation inhibitors are present in a fermentation medium in which a 7- chloretetracycline producing strain of S. aureofacien: is used.

Additional disclosures of the production of 7-chloro-6- demethyltetracycline by cultivating 7-chlortetracycline producing strains of S. aureofaciens in the presence of 6- methylation inhibitors are shown in the Goodman et al. U.S. Pat. Nos. 3,019,172 and 3,l27,328. These processes are also faced with the problem of concomitant formation of other tetracyclines. Thus, in each a mixture of 7-chlortetracycline, tetracycline, 7-chloro-6-demethyltetracycline and 6- demethyltetracycline is produced.

The concomitant production of o-demethyltetracycline (or other tetracyclines) is objectionable when 7-chloro-6- demethyltetracycline is the principal product being sought. While the specifications of pharmaceutical quality 7-chloro-6- demethyltetracycline permit small quantities of 6-dernethyltetracycline to be present, the presence of any sizable quantity of 6-demethyltetracycline is objectionable. The presence of these two antibiotics in the same fermentation mash in any sizable amounts also involves difficult problems of separation in the refining or extraction procedures when 7-chloro-6- demethyltetracycline is the desired product. it is possible, of course, to extract the two antibiotics from the fermentation mash and by selective refining procedures to effect a separation of the antibiotics. However, the refining procedures for effecting separation of the antibiotics are difficult and usually involve some loss in total antibiotic potency. Moreover, in those instances where 7-chloro-6-demethyltetracycline is the principal product of the fermentation, G-demethyltetracycline is unwanted and is customarily removed and discarded. Hence, any method that results in the production of 7-chloro- 6-demethyltetracycline to the exclusion of 6-demethyltetracycline constitutes a marked contribution to the art of antibiotic fermentation.

it is indeed surprising that the novel mutant strains of Streptomyces aureofaciens of the present invention will produce, in a conventional fermentation, 7-chloro-6- demethyltetracycline to the exclusion of o-demethyltetracycline. Furthermore, there is no detectable production of tetracycline or 7-chlorotetracycline. The discovery of these strains obviates the need for costly and difiicult extraction and purification procedures, since the crystals of 7-chloro-6- demethyltetracycline obtained are of pharmaceutical quality. Obviously the discovery of these strains denotes a marked contribution to the art of producing the highly effective antibiotic 7-chloro-6--demethyltetracycline.

In general, these new strains and the strains which are presently known in the art which produce 7-chloro-6- demethyltetracycline in conventional media may be divided in three categories: (i) S. aureofaciens strains ATCC l255l, 12552, 12553 and 12554 which are described in U.S. Pat. No. 2,878,289 McCormick et al.) and which produce varying amounts of 7-chloro-6-demethyltetracycline, 6-demethyltetracycline, tetracycline and 7chlorotetracycline; (2) S. aureofaciens strains V-l38, E-475, E-l3l1, ED-l723, ED-2047 and ED-23l4 which are disclosed in the following patents: V-l38 in U.S. Pat. No. 3,037,916 (Goodman), E-475, E-l3ll, ED-1 723 and [ED-2047 in U.S. Pat. No. 3,050,446 (Goodman), [ED-2314 in U.S. Pat. No. 3,l45,l54 (Goodman); which produce varying amounts of 7-chloro-6- demethyltetracycline and fi-demethyltetracycline to the virtual exclusion of methylated tetracyclines; and (3) the new strains of the present invention, e.g., -2322 and lE-2750, which produce 7-chloro-6-demethyltetracycline to the exclusion of all other tetracyclines.

The novel mutant strains of the present invention may be distinguished from the two groups of prior art strains by a mathematical interpretation of the spectrophotometric reflectance curves of their respective whole harvest mashes. More precisely, when the percent reflectance of diluted sam- 3 ,616,240 3 4 pics (1:200 aiu'usybr in? naoih sr'vesfinanie'ar H @662 potencyoftheferrnentathih mash, as may be convenyield (such as the examples set forth hereinafter} of the novel tionally determined, ceases to rise appreciably after the ferstrains of the present invention is plotted linearly against the mentation has proceeded for from about M to about 160 wavelength, the resulting curve, between 460 my and 520 mu, hours M 7 7 I i n M, A V v I I 7 I u will either exhibit a maximum or have a point of inflection of 5 V Th novel mutant strains f s aureofadens f {Hg present i zero slope. vention which produce no other tetracycline except 7-chloro- FIGS. 1 through 12 of the drawings are spectrophotometric fi-demethyltetracycline possess essentially the same reflectance curves obtained when l-centimeter glass cells are morphological characteristics as do the strains which con-. fi d with a diluted whole harvest mash sample which comitantly produce 7-chlorotetracycline, tetracycline, 7-

is taken from th har st rm ntati n r th Of the novel mllchloro--demethyltetracycline and -demethyltetracycline tant strains of the present invention as well as the additional and those strains which produce 7-chloro-6-demethyltetral0 previously-described priorart strains listed in table I. The cycline and a substantial proportion of -demethyltetraspectrophotometric reflectance curve of a material constitutes cycline, but differ among themselves in the same general a permanent record that does not require maintenance of a 15 manner that these prior art strains differ from each other as sample. Furthermore, the units in which the curve is expressed has been described in a number of scientific papers which are internationally understood and accepted as a means of have been published. The data appearing below will serve to measuring color. in FIGS. 1 through 12, the wavelength of .further distinguish the novel mutant strains of S. aureofaciens light in millimicrons is plotted as the abscissa against the from the original A-377 strain available as NRRL 2209 and reflectance as the ordinate. The wavelength of light has been the McCormick et al. strain, ATCC No. 12551 (8-604). in the adopted internationally as the fundamental standard of length following color observations on a variety of media, the color to which all other standards of length are referred. These specdata strain S-604 (ATCC l255l) have been reproduced from trophotometric reflectance curves were determined with a Pat.No.Z 78,2$2.

recording spectrophotometer known as the General El trie To illustrate the visual color variations among the novel mul-lardy Recording Spectrophotometer with the sample backe tant strains S. aureofaciens of the present invention, these with magnesium oxide, the reference standard being magnesi-f strains were grown on AP4 agar and the following observaum Oxide. a with he specular omeones 39991 35, ,tion nsrenaie--. V

COLOR OBSERVATIONS: l S. aureofact'ens; AP4 AGAR; SIX DAY INCUBATION AT 265 0.

Single colonies Strain Major Colony Type I Minor Colony Type 11 Mass growth 1E-2322 Burgundy to dark rose brown.-.. Ebony brown Burgundy to dark rose brown.

115-2750 Chocolate brown -do Rose brown.

1 Colors here and elsewhere throughout this specification are according to the Color Harmony Manual, Third Edition, Container Corporation of America.

curves are recorded on General Electric Co. Chart paper NP j FORMULATION OF AP4 AGAR 62581.

' With regard to the aforementioned spectrophotometric v determination it should be noted that dilution of the whole 40 I' harvest mash is necessary to produce the mathematical results zff' 32 53: as stated. if the whole harvest mash is used undiluted, the (Mann-rm, 0.2 lrlt'l'll stated reflectance curve will not be evident. it is only when the m whole harvest mash is diluted with water at the rate of one part s: 32m of whole harvest mash to 199 parts of water that the unusual i m n p" a results of a reflectance curve having either a maximum or a point of inflection of zero slope between 460 my and 520 my.

becomes evident. V W V W W m g V MW The novel S. aureofaciens strains of the present invention A whole harvest mash is the untreated mash obtained after a were differentiated from S. aureofaciens A-377 (NRRL 2209) fermentation employing the following medi n m by observation of growth characteristics on various media incubated at 26.5 C. until maturity.

Starch-45 grams c co 10 5 grams (1) GLYCEROL ASPARAGINE BEEF EXTRACT AGAR Nl'LCll .5 grams Cottonseed flour-45 grams 55 Yeastl .5 grams glycerol L0 grams -asparagrne 0.05 grams Waterq.s. to l 000 ml. Beei'extract 0.2 grams Lard oil-3% (Vol/vol. of medium) KHJ 0.05 grams I V WH."4 -AV A 1 w 7*;- i A Y 89C? Baal L5 IKIIIIS has proceeded to the point where biosynthesis of the primary 1 Adjust to pH 1.0 with KOH antibiotic product has stopped for all practical purposes. pommmmion Generally, under the conditions set forth hereinbelow. the an- Streplomyces aureojaciens Strain lid-2322 Strain 8-604 Growth Moderate, chocolate to rose brown Abundant, venetian red. Aerial hyphae Abundant, white becoming pussywillow grey. Abundant, white to rose grey. Sporulation Incomplete Slight becoming abundant. Dlfiusible pigment Deep amber Refinish-brawn. Boverse Deep brown to rose brown Brown-mahogany.

7 Strain .A-377 Strain 113-2750 Growth Fair Moderate, sepia brown to deep brown mahogany. Aerial hyphae-... White, uniform... Thinto fair, white. S rulation None None. Dlfluslble pigment. Yellow Tawny becoming amber. Reverse Yellow to light orange yellow Sepia brown to deep brown mahogany- (2) DEXTRIN CZAPEK-DOX AGAR (4) Q4 CORNSTEEP AGAR Dextrin 1.0 grams NaNO: 0.2 grams Cornsteep 9 grams K,HPO. 0.l gra 5 Sucrose 10 grams MgSQ .7H,O 0.05 grams MgSO,.7l-i,0 0.25 grams KCl 0.05 grams (NH,),HPO, 2 grams FeSO .7H,O 0001 grams KH,P0, 4 grams Bacto agar 1.5 grams Crude agar 30 grams Distilled water, q.s. 100.0 ml. Water s 1000 mi Post Sterilization pH is 7.2 10 Post sterilization pH is 6.5

Streptomyces aureofacz'ens Strain 1E-2322 Strain 8-604 Growth Aenal hyphae. Sporulation Difiusible pigment Do. Reverse Apigmentous.

Strain a-arr Strain 113-2750 Growth Profuse Confluent, thin, transparent, colorless. Aenal hyphae. Abundant, lead grey, water white surface globules. one. Sporulation Abun ant Do. Diffusible pigmen Slight, pale yellow Do. Reverse Apigmentous Translucent (3) AP4 AGAR (AS DESCRIBED ABOVE) Streptomyccs aurcofaciens Strain 113-2322 Strain 8-604 Growth Profuse, burgundy to dark rose brown Profuse. Aerial hyphae. oderate, pussywillow grey to ashes Abundant, dark rose taupe. Sporulation Fair Very abundant, uniform. Difiusible pigmcn Deep red-brown Very concentrated, deep brown to deep brown mahogany.

Reverse Burgundy to dark rose brown Dark brown mahogany.

Strain A-377 Strain 113-2750 Growth Profuse Abundant to profuse, cordovan to ebony brown. e ial hyphae Abundant, beaver Thin to moderate, pussywillow grey. Sporulation Very abundant, uniform Thin to moderate. Difiusible pigment. Light greenish-yellow Reddish brown to deep reddish brown. Reverse Covert brown Cordovan to ebony brown.

Streptomyces aureofaciens Strain 1E-2322 Strain S-604 Profuse, burgundy. Profuse, chocolate.

Profuse. Burgundy. Do.

Strain 112-2750 Growth Excellent, pale yellow Abundant, cordovan to ebony brown. Aerial hyphae Profuse, dark brown Fair to moderate, pussywillow grey to ashes. Sporulation Profuse Thin to fair. Difiusible pigment. Orange-brown Cordovan. Reverse Orange to orange-yellow Ebony brown.

(5) OTHER AGAR MEDIA Streptomyccs aureofacicns Medium Strain lE-2322 Strain 8-604 u ient agar Thin growth; chocolate brown. No aerial liypliae. Poor growth. Taupe brown to dark ro rh o m Light purplish soluble pigment. Reverse: chocolate hyphae. Reverse: taupe brown. Reddish brown brown. soluble pigment.

Glucose asparagine-meat extract agar. Abundant growth; deep brown. Abundant aerial hy- Abundant growth. Heavy aerial hyphac mottled. Rose phae; white becoming ashes. Abundant spornlation. taupe to fawn to camel. Sporulation: abundant. Re- Deep yellow soluble pigment. Reverse: deep brown. verse: taupe brown. Reddish brown soluble pigment. P mp1s milk o m m d growth collar; taupe brown to rose brown. Slight deep red mahogany growth collar. Little signifi- Little significant pH change. False alkaline color cant pH change nor apparent peptonization. Slight. reaction due to pigment diffusion. pH 6.75 at 2 false alkaline color reaction due to diffusion of soluble W06 pigment.

Potato S ts Profuse. moist crenulated growth; dark luggage tan Profuse, moist smooth nodulatctl gro th; d brown becoming 7po 1 to black plum. Rose taupe soluble mahogany with peach tan trace. Aerial hyphae; none pigment. No aerial hyphae. to abundant becoming white to camel. Sporulation abundant in areas of heavy aerial hyphae. Chocolate. to chocolate brown soluble pigment.

1 Color chip code according to the Color Harmony Manual, Third Edition, Container Corp. of America. No assigned color name.

Streptomyces aureofacicns Medium Strain A-377 Strain 1E-2750 Nutrient agar. Fair growth. N0 aerial hyphae. Reverse: pale yellow. Poor to fair confluent growthgdark brown mahogany. No aerial Yellow to light brownish yellow soluble pigment. hyphae. Tawny diffusible pigment tinted reddish brown. Re-

verse: dark brown mahogany. Glucose asparagiue- Fair growth. Aerial hyphae white becoming increasingly Good to abundant growth; copper brown to brown mahogany. meat extract agar. grey with increase in spore formation. Reverse: light Abundant aerial llyphae uniformly pussywillow grey. Abundant yellow. Light yellow soluble pigment. sporulation. Diilusible pigment light tawny-yellow becoming amber. Reverse: copper brown to brown mahogany becoming dark brown mahogany.

Purple milk Slight white to pale yellow growth collar. Little significant Pronounced growth collar; taupe brown to dark rose brown. Little pH change nor apparent peptonization in 15 days. significant plI change. False alkaline color reaction due to pigment diffusion. pH 6.75 at 2 weeks. Potato slants Profuse, moist smooth nodulated growth; light melon yel- Profuse, moist, crenulated growth; light fawn to copper brown below to antique rose. Aerial hyphae trace. No soluble pi coming black plum. Tendency to form white dense areas of aerial ment. hyphae at separated foei. Diffusible pigment; rose taupe to taupe brown.

(6) MICROSCOPIC OBSERVATIONS S. aureofaciens Glucose asparagine beef extract agar Q4 agar Strain 115-2322 Mycelium Flfzguous, continuous, branched. Diameter 0.8 to Flfxuous, continuous, branched. Diameter 0.8 to .44. .Zn. Spores Spherical to ovoidal. Diameter 0.8 to 1.5 4. Spherical to ovoidal. Diameter 0.8 to 1.5,. Strain 8-604 Mycellum... Fleruous, continuous, branched. Diameter 08 to Flexuous, continuous, branched. Diameter 0.8 to p. 1.2 Spores spheroidal to ovoidal. Diameter 1.5 to 2.0;1. Spheroidal to ovoidal. Diameter 1.5 to 2.0,. Strain A-377 Mycelium Flexuous, continuous, branched. Diameter 0.7 to Flexuous, continuous, branched. Diameter 0.8 to

. pt. Spores spheroidal to ovoidal. Diameter 1.5 to 2.0

. Flexuous, continuous, branched. Diameter 0.8 to

Strain lE-2750 Mycelium.

1.2a. Spores Spherical to ovoidal. Diameter 0.8 to 1.5;.

Noun-lilycelial and spore morphology, as viewed through the light microscope, is ap arently similar for strains 113-2322 and 1153-2750 as for strains A-377 an 8-604. However, the diameters for typical mycelium and for spores of the former strains appear to be characteristically smaller The conditions (if the fEm entation are generalTy :E'm? as for the presently known methods of producing chlorotetracycline by fermentation. That is, the fermentation medium contains the usual assimilable sources of carbon, nitrogen and inorganic salts. Suitable nutrient substances include starch, dextrose, cane sugar, glucose, molasses, soybean meal, milk solids, yeast, meat extracts, peptone, urea, cornsteep liquor,

cottonseed meal, distillers-3 solubles, glyceride oils, fish meal and other conventional substances. The inorganic salts include such things as calcium carbonate, ammonium sulfate, ammonium chloride, and salts of the various trace elements such as manganese, cobalt, zinc, copper, iron and the like.

The other general conditions of the fermentation such as hydrogen ion concentration, temperature, time, rate of aeration, preparation of the inoculum, sterilization, inoculation and the like are conventional and may be similar to those for the production of 7-chloro-6-demethyltctracycline shown in US. Pat. No. 2,878,289 (McCormick et a1.).

1.2;. spheroidal to ovoidal. Diameter 1.5 to 2.0 Flleiuous, continuous, branched. Diameter 08 to n. Spherical to ovoidal. Diameter 0.8 to 1.5

than for the latter. All strains demonstrate continuous, ilexuous, branched mycelium with an occasional tendency of aerial hyphae to develop books or loops, or to coil into primitive loose spirals as is typical of the species S. aureofacie'rls.

8-1071 (ATCC 12552) V-62 (ATCC 12553) B-740 (ATCC 12554) ED-l 723 lid-2314 lE-2322 (NRRL 3204) 113-2750 (NRRL 3234) All of the flasks were then incubated on a rotary shaker with a ZVz-inch circular motion at 185-200 r.p.m. at 25 C. for 180 hours. Samples were withdrawn, diluted with 199 parts of water, and the reflectance curves determined as set forth hereinabove. The results are set forth in FIGS. 1 through 12. The harvest mash assays are recorded in table I below. These assays were performed using the following methods:

1 No G-demethyltctracycline was found at the lowest detectable level 01 1 /1111.

The recovery of the 7-chloro-6-demethyltetracycline from the fermentation liquor is conventional and need not be described, as numerous methods for the recovery of this product from fermentation liquors have already been published.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 A fermentation medium was prepared which contained the following ingredients:

Stach-4S grams CaCO 10.5 grams NH,C11.5 grams Cottonseed flour-45 grams Yeast1.5 grams Waterq.s. to- 1000 milliliters This medium was dispensed in appropriate amounts into flasks containing 3 percent (vol/vol. of medium) of lard oil and then sterilized. These flasks were then divided into 12 approximately equal groups. Each group was then inoculated with a vegetative inoculum of one of the following strains of S. aureofaciens.

Assay No. 1

This assay is based upon the difference in the rates of conversion of 7-chlore-6-demethyltetracycline and 6-demethyltetracycline to their respective anhydro forms by an acid catalyzed, first order reaction. The degree of conversion to the anhydro form depends upon time, temperature and acid strength involving a reaction which results in a decrease in absorbance at 368 my and an increase in absorbance at 440 mu.

Assay No. 2

This assay is based upon the same basic principle as Assay N o. 1 except that in this case the sample is treated with acid to degrade all tetracycline present to the anhydro form in one instance and compared to a sample treated with water in the second instance.

Assay No. 3

The assay figures in this column were determined by a conventional paper chromatographic procedure using the system 0.3 M sodium phosphate n-butyl acetate, giving an R, of 0.30 for G-demethyltetracycline. Three similar systems, published in the Journal of the American Chemical Society, Vol. -79, pp. 4561-4563 1957), McCormick et al.) are listed below.

System -Demethyltetracycline 03 M Sodium Phosphate/nbutanol (pH 30) 0.30 Mac llvaines Buffer/ethyl acetate (pH in this table I, the percent 6-demethyltetracycline (Column 4) of the first four strains (8-604, 8-1071, V-62 and B 740) was determined by finding the amount of 6-demethyltetracycline by paper chromatography (Column 3) and dividing this figure by the total amount of demethyltetracyclines determined by spectrophotometric analysis (Column 1) plus the chromatographic analysis (Column 3). The percent -demethyltetracycline (Column 4) produced by the balance of the strains is determined by dividing the chromatographic analysis (Column 3) by the spectrophotometric analysis of the total tetracyclines (Column 22 What is claimed is:

l. The process of producing 7-chloro-6-demethyltetracycline which comprises cultivating a strain of Streptomyces aureofaciens which produces 7-chl0ro-6-demethyltetracycline exclusively selected from the group consisting of S. aureofaciens NRRL 3204 and mutants thereof under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts until substantial quantities of 7-chloro-6-demethyltetracycline as produced, said 7-chloro-6-demethyltetracyclinc-producing strain of Streptomyces aureofaciens being characterized by its ability to impart to a ZOO-fold aqueous dilution of the whole harvest mash a color characterized by a spectrophotometric reflectance curve, when plotted linearly, having a maximum between 460 my. and 520 mu.

2. The process of producing 7-chloro-6-demethyltetracycline which comprises cultivating a strain of Streptomyces aureofaciens which produces 7-chloro-6-demethyltetracycline exclusively selected from the group consisting of S. aureofaciens NRRL 3234 and mutants thereof under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts until substantial quantities 7-chloro-6-demethyltetracycline are produced, said 7-chloro-6-demethyltetracycline-producing strain of Streptomyces aureofaciens being characterized by its ability to impart to a ZOO-fold aqueous dilution of the whole harvest mash a color characterized by a spectrophotometrie reflectance curve, when plotted linearly, having a point of inflection of zero slope between 460 my and 520 mu. 

2. The process of producing 7-chloro-6-demethyltetracycline which comprises cultivating a strain of Streptomyces aureofaciens which produces 7-chloro-6-demethyltetracycline exclusively selected from the group consisting of S. aureofaciens NRRL 3234 and mutants thereof under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts until substantial quantities of 7-chloro-6-demethyltetracycline are produced, said 7-chloro-6-demethyltetracycline-producing strain of Streptomyces aureofaciens being characterized by its ability to impart to a 200-fold aqueous dilution of the whole harvest mash a color characterized by a spectrophotometric reflectance curve, when plotted linearly, having a point of inflection of zero slope between 460 m Mu and 520 m Mu . 